Phosphate rebinding induces force reversal via slow backward cycling of cross-bridges.

OBJECTIVE: Previous studies on muscle fibers, myofibrils, and myosin revealed that the release of inorganic phosphate (P(i)) and the force-generating step(s) are reversible, with cross-bridges also cycling backward through these steps by reversing force-generating steps and rebinding P(i). The aim was to explore the significance of force redevelopment kinetics (rate constant k (TR)) in cardiac myofibrils for the coupling between the P(i) binding induced force reversal and the rate-limiting transition f (-) for backward cycling of cross-bridges from force-generating to non-force-generating states. METHODS: k (TR) and force generation of cardiac myofibrils from guinea pigs were investigated at 0.015-20 mM P(i). The observed force-[P(i)], force-log [P(i)], k (TR)-[P(i)], and k (TR)-force relations were assessed with various single-pathway models of the cross-bridge cycle that differed in sequence and kinetics of reversible P(i) release, reversible force-generating step and reversible rate-limiting transition. Based on the interpretation that k (TR) reflects the sum of rate-limiting transitions in the cross-bridge cycle, an indicator, the coupling strength, was defined to quantify the contribution of P(i) binding induced force reversal to the rate-limiting transition f (-) from the [P(i)]-modulated k (TR)-force relation. RESULTS: Increasing [P(i)] decreased force by a bi-linear force-log [P(i)] relation, increased k (TR) in a slightly downward curved dependence with [P(i)], and altered k (TR) almost reciprocally to force reflected by the k (TR)-force relation. Force-[P(i)] and force-log [P(i)] relations provided less selectivity for the exclusion of models than the k (TR)-[P(i)] and k (TR)-force relations. The k (TR)-force relation observed in experiments with cardiac myofibrils yielded the coupling strength +0.84 ± 0.08 close to 1, the maximum coupling strength expected for the reciprocal k (TR)-force relationship. Single pathway models consisting of fast reversible force generation before or after rapid reversible P(i) release failed to describe the observed k (TR)-force relation. Single pathway models consistent with the observed k (TR)-force relation had either slow P(i) binding or slow force reversal, i.e., in the consistent single pathway models, f (-) was assigned to the rate of either P(i) binding or force reversal. CONCLUSION: Backward flux of cross-bridges from force-generating to non-force-generating states is limited by the rates of P(i) binding or force reversal ruling out other rate-limiting steps uncoupled from P(i) binding induced force reversal.